BACKGROUND: African swine fever (ASF) is a highly contagious and severe haemorrhagic disease of Suidae, with mortalities that approach 100 percent. Several studies suggested the potential implication of non-biting dipterans in the spread of ASFV in pig farms due to the identification of the ASFV DNA. However, to our knowledge, no study has evaluated the viral DNA load in non-biting dipterans collected in outbreak farms and no risk factors have been analysed. In this context, our study aimed to analyse the risk factors associated with the presence of non-biting dipterans collected from ASF outbreaks in relation to the presence and load of viral DNA.
METHODS: Backyard farms (BF), type A farms (TAF), and commercial farms (CF), were targeted for sampling in 2020. In 2021, no BF were sampled. Each farm was sampled only once. The identification of the collected flies to family, genus, or species level was performed based on morphological characteristics using specific keys and descriptions. Pools were made prior to DNA extraction. All extracted DNA was tested for the presence of the ASFV using a real-time PCR protocol. For this study, we considered every sample with a CT value of 40 as positive. The statistical analysis was performed using Epi Info 7 software (CDC, USA).
RESULTS: All collected non-biting flies belonged to five families: Calliphoridae, Sarcophagidae, Fanniidae, Drosophilidae, and Muscidae. Of the 361 pools, 201 were positive for the presence of ASFV DNA. The obtained CT values of the positive samples ranged from 21.54 to 39.63, with a median value of 33.59 and a mean value of 33.56. Significantly lower CT values (corresponding to higher viral DNA load) were obtained in Sarcophagidae, with a mean value of 32.56; a significantly higher number of positive pools were noticed in August, mean value = 33.12.
CONCLUSIONS: Our study brings compelling evidence of the presence of the most common synanthropic flies near domestic pig farms carrying ASFV DNA, highlighting the importance of strengthening the biosecurity measures and protocols for prevention of the insect life cycle and distribution.

African swine fever (ASF) is a severe viral disease characterized by high lethality in suids and caused by the African Swine Fever Virus (ASFV). The ASF genotype I virus was introduced to Europe in 1957, marking the onset of the first European epidemic wave. In 2007, ASFV genotype II was detected in Georgia, affecting domestic pigs and wild boars before spreading to various European and extra-European countries, including Italy. The first case of ASFV in Italy was documented on 7 January 2022, in a wild boar in the Piedmont region. Since then, several ASFV-positive wild boar carcasses have been identified in the Piedmont and Liguria regions. By June 2023, ASFV had spread to Lombardy, one of the major pig-producing regions in northern Italy; the virus was first detected in early summer in wild boar carcasses. Two months later, it was diagnosed in a commercial pig farm as a consequence of the disease\'s spread amongst wild boars and an increase in the viral environmental load. This report aims to describe the features of ASFV domestic pig outbreaks that occurred in the Zinasco municipality (Lombardy) and the joint efforts to mitigate potential direct and indirect economic impacts on the Italian and global pig industry. The epidemiological investigation and the measures implemented, which were all performed according to national and European regulations, as well as exceptional ad hoc measures aimed at protecting the pig industry, are described in order to provide a practical and effective approach to combating ASF.

Candidate vaccines against African swine fever virus (ASFV) based on naturally attenuated or genetically modified viruses have the potential to generate protective immune responses, although there is no consensus on what defines a protective immune response against ASFV. Studies, especially in sensitive host species and focused on unravelling protective mechanisms, will contribute to the development of safer and more effective vaccines. The present study provides a detailed analysis of phenotypic and functional data on cellular responses induced by intradermal immunization and subsequent boosting of domestic pigs with the naturally attenuated field strain Lv17/WB/Rie1, as well as the mechanisms underlying protection against intramuscular challenge with the virulent genotype II Armenia/07 strain. The transient increase in IL-8 and IL-10 in serum observed after immunization might be correlated with survival. Protection was also associated with a robust ASFV-specific polyfunctional memory T-cell response, where CD4CD8 and CD8 T cells were identified as the main cellular sources of virus-specific IFNγ and TNFα. In parallel with the cytokine response, these T-cell subsets also showed specific cytotoxic activity as evidenced by the increased expression of the CD107a degranulation marker. Along with virus-specific multifunctional CD4CD8 and CD8 T-cell responses, the increased levels of antigen experienced in cytotoxic CD4 T cells observed after the challenge in immunized pigs might also contribute to controlling virulent infection by killing mechanisms targeting infected antigen-presenting cells. Future studies should elucidate whether the memory T-cell responses evidenced in the present study persist and provide long-term protection against further ASFV infections.

The Postmortem Interval (PMI) is the time from the death of an animal to its discovery. From a veterinary forensic standpoint, an accurate estimation of the PMI is of particular importance, especially with the observed increase in deaths of domestic and wild animals. A preliminary study was conducted using the eyes of domestic pigs. A biochemical analysis was conducted on the vitreous humor of the eye, whilst a histological analysis was conducted on the retina. The eyes were stored at +4 °C and changes were assessed at time intervals of 0, 12, 24, 48, and 120 h. The biochemical analysis during the PMI established a decrease in sodium, chlorine, and glucose concentrations, and a rise in potassium concentration. Accordingly, a simple linear regression showed a significant correlation between changes in concentrations of sodium (Na+), potassium (K+), chloride (Cl-), and glucose, in relation to the PMI. The histological analysis showed evident morphological changes in the retina, which included homogenization of the rod and cone cells, pyknosis of the outer nuclear layer, homogenization of the outer plexiform layer, pyknosis of the inner nuclear layer, homogenization of the inner plexiform layer, and pyknosis of the nuclei of the ganglion layer of the retina.

Soil-transmitted helminth (STH) infections are caused by roundworms, hookworms, whipworms, and thread worms. Accurate diagnosis is essential for effective treatment, prevention, and control of these infections. This study evaluates a new diagnostic method called Single-image Parasite Quantification (SIMPAQ), which uses a lab-on-a-disc (LoD) technique to isolate STH eggs into a single imaging zone for digital analysis. The study evaluates the purification performance of the SIMPAQ technique for detecting STH eggs in animal samples. This was a cross-sectional study conducted among 237 pigs and 281 dogs in the Morogoro region in Tanzania. Faecal samples were collected and processed with the LoD technique, as well as flotation and McMaster (McM) methods for comparison purposes. The overall prevalence of STH infections was high as per the LoD technique (74%), followed by McM (65.44%) and flotation (65.04%). Moreover, the overall performance of the LoD technique, using McM as the gold standard, was 93.51% (sensitivity), 60.89% (specificity), 81.91% (PPV), and 83.21% (NPV). The LoD technique exhibited high prevalence, sensitivity, and NPV, which demonstrates its value for STH egg detection and its crucial role in the era of accurate STH diagnosis, promoting proper management of the infection.

UNASSIGNED: African swine fever (ASF) is a notifiable disease of swine that impacts global pork trade and food security. In several countries across the globe, the disease persists in wild boar (WB) populations sympatric to domestic pig (DP) operations, with continued detections in both sectors. While there is evidence of spillover and spillback between the sectors, the frequency of occurrence and relative importance of different risk factors for transmission at the wildlife-livestock interface remain unclear.
UNASSIGNED: To address this gap, we leveraged ASF surveillance data from WB and DP across Eastern Poland from 2014-2019 in an analysis that quantified the relative importance of different risk factors for explaining variation in each of the ASF surveillance data from WB and DP.
UNASSIGNED: ASF prevalence exhibited different seasonal trends across the sectors: apparent prevalence was much higher in summer (84% of detections) in DP, but more consistent throughout the year in WB (highest in winter with 45%, lowest in summer at 15%). Only 21.8% of DP-positive surveillance data included surveillance in WB nearby (within 5 km of the grid cell within the last 4 weeks), while 41.9% of WB-positive surveillance samples included any DP surveillance samples nearby. Thus, the surveillance design afforded twice as much opportunity to find DP-positive samples in the recent vicinity of WB-positive samples compared to the opposite, yet the rate of positive WB samples in the recent vicinity of a positive DP sample was 48 times as likely than the rate of positive DP samples in the recent vicinity of a positive WB sample. Our machine learning analyses found that positive samples in WB were predicted by WB-related risk factors, but not to DP-related risk factors. In contrast, WB risk factors were important for predicting detections in DP on a few spatial and temporal scales of data aggregation.
UNASSIGNED: Our results highlight that spillover from WB to DP might be more frequent than the reverse, but that the structure of current surveillance systems challenge quantification of spillover frequency and risk factors. Our results emphasize the importance of, and provide guidance for, improving cross-sector surveillance designs.

African swine fever virus (ASFV) is the etiological agent of a haemorrhagic disease that threatens the global pig industry. There is an urgency to develop a safe and efficient vaccine, but the knowledge of the immune-pathogenetic mechanisms behind ASFV infection is still very limited. In this paper, we evaluated the haematological and immunological parameters of domestic pigs vaccinated with the ASFV Lv17/WB/Rie1 strain or its derived mutant Lv17/WB/Rie1/d110-11L and then challenged with virulent Armenia/07 ASFV. Circulating levels of C-reactive protein (CRP), 13 key cytokines and 11 haematological parameters were evaluated throughout the study. Lv17/WB/Rie1 triggered an inflammatory response, with increased levels of CRP and pro-inflammatory cytokines, and induced lymphopenia, thrombocytopenia and a decline in red blood cell (RBC) parameters, although this was transitory. Lv17/WB/Rie1/d110-11L triggered only transitory thrombocytopenia and a mild inflammatory reaction, with no increase in serum levels of pro-inflammatory cytokines, but it raised IL-1Ra levels. Both strains counteracted several adverse reactions elicited by virulent challenge, like thrombocytopenia, a decline in RBC parameters, and inflammation. Within this paper, we provided a deep portrayal of the impact of diverse ASFV strains on the domestic pig\'s immune system. A better understanding of these immune-pathological mechanisms would help to design suitable vaccines against this disease.

Smallholder subsistence pig production is common in Uganda and African swine fever (ASF) is endemic in the country, with its spread driven by human activities along the smallholder value chain. Previous research in the study area has revealed that many stakeholders are aware of how ASF is spread, its prevention and control, and have a generally positive attitude towards biosecurity. Despite this, even basic biosecurity is largely lacking. Costs, as well as a lack of adaptation to the local context, culture and traditions have been identified as factors hindering biosecurity implementation. Community engagement and local ownership of disease problems are increasingly recognised as important for improving disease prevention and control. The objective of this study was to investigate the capacity of participatory action at community level with broad inclusion of stakeholders to improve biosecurity in the smallholder pig value chain. Specific attention was paid to participants\' perceptions and experiences of implementing the biosecurity measures included in their co-created community contracts. The study was conducted in Northern Uganda in villages purposively selected on the basis of previous occurrences of ASF. In each village, farmers and traders were also purposively selected. At a first meeting, basic information about ASF was shared and participants presented with a list of biosecurity measures adapted for farmers and traders respectively. Participants discussed each measure in farmer and trader subgroups, decided on the measures to implement for one year, and signed a community contract to this effect. The following year, interviews were again undertaken and implementation support given. Interview data were coded and thematically analysed. Each subgroup chose a minimum of three and a maximum of nine measures, with wide variations between villages in their selection of measures. At the follow-ups, none of the subgroups had fully implemented what had been agreed in their contract, but all had changed some of their biosecurity routines. Some frequently recommended biosecurity measures, such as not borrowing breeding boars, were not considered feasible. Relatively simple and cheap biosecurity measures were rejected for reasons of cost, highlighting the participants\' general level of poverty and the relevance of poverty as a specific factor governing disease control results. The participatory methodology allowing for discussions, co-creation and the option to refuse measures seemed to facilitate the implementation of measures that had initially been thought to be controversial. The broad community approach was deemed to be positive for strengthening community identity, cooperation and implementation.

Swine influenza A virus (swIAV) plays an important role in porcine respiratory infections. In addition to its ability to cause severe disease by itself, it is important in the multietiological porcine respiratory disease complex. Still, to date, no comprehensive diagnostics with which to study polymicrobial infections in detail have been offered. Hence, veterinary practitioners rely on monospecific and costly diagnostics, such as Reverse Transcription quantitative PCR (RT-qPCR), antigen detection, and serology. This prevents the proper understanding of the entire disease context, thereby hampering effective preventive and therapeutic actions. A new, nanopore-based, metagenomic diagnostic platform was applied to study viral and bacterial profiles across 4 age groups on 25 endemic swIAV-infected German farms with respiratory distress in the nursery. Farms were screened for swIAV using RT-qPCR on nasal and tracheobronchial swabs (TBS). TBS samples were pooled per age, prior to metagenomic characterization. The resulting data showed a correlation between the swIAV loads and the normalized reads, supporting a (semi-)quantitative interpretation of the metagenomic data. Interestingly, an in-depth characterization using beta diversity and PERMANOVA analyses allowed for the observation of an age-dependent interplay of known microbial agents. Also, lesser-known microbes, such as porcine polyoma, parainfluenza, and hemagglutinating encephalomyelitis viruses, were observed. Analyses of swIAV incidence and clinical signs showed differing microbial communities, highlighting age-specific observations of various microbes in porcine respiratory disease. In conclusion, nanopore metagenomics were shown to enable a panoramic view on viral and bacterial profiles as well as putative pathogen dynamics in endemic swIAV-infected herds. The results also highlighted the need for better insights into lesser studied agents that are potentially associated with porcine respiratory disease. IMPORTANCE To date, no comprehensive diagnostics for the study of polymicrobial infections that are associated with porcine respiratory disease have been offered. This precludes the proper understanding of the entire disease landscape, thereby hampering effective preventive and therapeutic actions. Compared to the often-costly diagnostic procedures that are applied for the diagnostics of porcine respiratory disease nowadays, a third-generation nanopore sequencing diagnostics workflow presents a cost-efficient and informative tool. This approach offers a panoramic view of microbial agents and contributes to the in-depth observation and characterization of viral and bacterial profiles within the respiratory disease context. While these data allow for the study of age-associated, swIAV-associated, and clinical symptom-associated observations, it also suggests that more effort should be put toward the investigation of coinfections and lesser-known pathogens (e.g., PHEV and PPIV), along with their potential roles in porcine respiratory disease. Overall, this approach will allow veterinary practitioners to tailor treatment and/or management changes on farms in a quicker, more complete, and cost-efficient way.

The first case of African swine fever in domestic pigs in Serbia was in 2019. The following year, the disease was confirmed in wild boar. Thenceforth, ASF has been continuously reported in both wild and domestic pigs. The outbreaks in domestic pigs could not be linked directly to wild boars, even though wild boars were endemically infected, and reservoirs for ASF. This study aimed to investigate outbreaks and routes of transmission in domestic pigs in a region of central Serbia where no outbreaks in wild boar were reported. Fourteen outbreaks of ASF on backyard farms with low biosecurity were traced back, and no connection to wild boar was found. The epidemic investigation covered 2094 holdings, with 24,368 pigs, out of which 1882 were tested for ASF. In surrounding hunting grounds, field searches were conducted. Dead wild boars were found, and 138 hunted wild boars were negative for ASFV. It was concluded that outbreaks in 2021 were provoked by the illegal trade of live animals and pig products. Even though infective pressure from wild boars is assumed, no positive cases have been found, while the ASFV spreads within the domestic swine population evidenced in four recent outbreaks in 2022.